Molecular-weight dependence of the glass transition temperature of freely-standing poly(methyl methacrylate) films.

We have used transmission ellipsometry to measure the glass transition temperature, T(g), of freely-standing films of atactic and syndiotactic poly(methyl methacrylate) (PMMA). We have prepared films with different molecular weights, MW, (159 x 10(3) < M (w) < 1.3 x 10(6)) and film thicknesses, h, ( 30 nm < h < 200 nm). For the high-MW ( M (w) > 509 x 10(3)) atactic PMMA films, we find that T(g) decreases linearly with decreasing h, which is qualitatively similar to previous results obtained for high-MW freely-standing polystyrene (PS) films. However, the overall magnitude of the T(g) reduction is much less (by roughly a factor of three) for the high-MW freely-standing PMMA films than for freely-standing PS films of comparable MW and h. The observed differences between the freely-standing PMMA and PS film data suggest that differences in chemical structure determine the magnitude of the T(g) reduction and we discuss the possible origins of these differences. Our analysis of the MW-dependence of the T(g) reductions suggests that the mechanism responsible for the MW-dependent T(g) reductions observed in the high-MW freely-standing films is different than that responsible for the MW-independent T(g) reductions observed in the low-MW freely-standing and supported films.

Expression of human chorionic gonadotropin uniformly labeled with NMR isotopes in Chinese hamster ovary cells: an advance toward rapid determination of glycoprotein structures.

Most secreted eukaryotic proteins are modified by glycosylation, and it has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carbohydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solved by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycosylated hCG is biologically inactive, the crystallographic structure confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature of interactions between the carbohydrate side chains and the protein backbone by crystallographic methods. Structural information via NMR techniques can be obtained from proteins in solution if they can be uniformly labeled with 13C and 15N isotopes. We report the first such uniform labeling of a glycoprotein using a universal 13C- and 15N-labeling medium to express 13C, 15N-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG as well as that of its free subunits. The 13C, 15N-labeled recombinant hCG and its separated subunits are shown to be nearly identical to urinary hCG reference preparations on the basis of protein chemical studies, immunochemistry, biological activity, and the capability of isolated hormone subunits to recombine to form biologically active hormone. Mass spectrometric analysis and preliminary NMR studies indicate that the isotopic labeling is uniform and greater than 90% after only two growth passages in the labeling media. One unexpected finding during subunit purification was that lyophilization of glycoproteins from trifluoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.

Isolation and characterization of human pituitary chorionic gonadotropin.

Human CG (hCG) is the hormone associated with the maintenance of pregnancy. Although three related glycoprotein hormones, LH, hFSH, and hTSH, are secreted by the pituitary, HCG is the only one of this family of glycoprotein hormones that is produced by the placenta in primates to maintain the steroid hormone secretions of the corpus luteum. Although hCG is not considered to be a pituitary hormone, hCG-like immunoreactive materials have been reported in pituitary tissue, blood, and urine from healthy nonpregnant individuals for 2 decades, but it was never isolated. We now report the purification and characterization of pituitary hCG from acetone-preserved human pituitary glands. After gell permeation chromatography, the fractions containing hCG molecules were pooled and purified by immunoaffinity chromatography using antibodies to the COOH-terminal region of hCGbeta. Amino acid analyses, amino-terminal sequence analyses, as well as mass spectrometric studies gave similar results for both pituitary hCG and hCG purified from the urine of pregnant women. Analyses for sulfate and sialic acid contents demonstrated that pituitary hCG contained both sulfate and sialic acid. In vitro biological activity of pituitary hCG indicated that it was 50% as active as hCG purified from the urine of pregnant women in cAMP assays.

The expression, characterization, and crystallization of wild-type and selenomethionyl human chorionic gonadotropin.

Although the glycoprotein hormone hCG was crystallized over 4 yr ago, it is only now that three-dimensional structural information is available. This manuscript reports the method for successful production of modified expressed hormone, the characteristics of the crystallized protein, and unexpected observations during the crystallization process. Two different routes of solution to the structure of hCG were followed. The first was based on the traditional method of heavy atom isomorphous replacement, and the second was the more novel method of expressing the protein with selenomethionine substituting for methionine and applying multiwavelength anomalous diffraction analysis. Selenomethionyl hCG was employed to successfully grow the crystals used for the solution of the structure of hCG after partial deglycosylation by hydrogen fluoride (HF) treatment. The selenomethionyl hCG proved to be more hydrophobic than the expressed form of native hCG. Furthermore, expressed forms of hCG that were deglycosylated by HF proved to be more intact and less susceptible to peptide bond cleavages during the crystallization process than the urinary form of HF-treated hCG studied previously. It was found that addition of reducing agent during the crystallization period was necessary for the growth of crystals of HF-treated selenomethionyl hCG suitable for diffraction studies. Growth of crystals of HF-treated expressed hCG were accelerated by the addition of dithiothreitol, but would successfully grow without reductant. HPLC analysis of the HF-treated hormones before and during the crystallization process was used to identify alterations in the molecules, including oxidation and aggregation, both of which may affect the growth of crystals.

Expression and DNA-binding properties of the 14K carboxyl terminal fragment of Escherichia coli DNA topoisomerase I.

DNA sequence coding for the last 121 amino acids of Escherichia coli topoisomerase I was synthesized by PCR and cloned into a plasmid under the control of the T7 promoter. Induction of T7 RNA polymerase in E. coli carrying the plasmid clone resulted in over-expression of this C-terminal domain fragment previously shown to confer higher DNA binding affinity to the enzyme. Purification to homogeneity was achieved by phosphocellulose and single-stranded DNA agaraose chromatography. Direct interaction between this 14K domain and poly(dA) was demonstrated by UV spectroscopy. Noncovalent complexes formed between this fragment and oligo(dT) 8 and oligo(dT) 16 can also be trapped by photo-crosslinking.

The efficacy of insecticides against the screw-worm fly (Chrysomya bezziana).

A number of insecticides used for ectoparasite control in the livestock industry were screened for their efficacy against larvae of the screw-worm fly, Chrysomya bezziana, using in vivo and laboratory tests. Proprietary screw-worm fly treatments (after exposure to outdoor conditions for up to 10 days) were also tested against eggs and adults of C bezziana. Three of these were also evaluated on naturally acquired screw-worm infestations. Residual protection was generally of short duration. Among the organophosphorus compounds, the most effective formulations contained relatively high concentrations (3 to 4% al) of coumaphos, 2.5% fenchlorphos or low concentrations (0.05 to 0.5% al) of diazinon, chlorfenvinphos and fenthion methyl. Two chlorinated hydrocarbon insecticides containing 3% lindane and 5% dieldrin were very effective but are now prohibited for use in Australia. Preparations had serious deficiencies when used under field conditions, especially for treating large, deepseated myiases for which systemic insecticides are recommended. A comparison of methods demonstrated that a laboratory test could supersede live animal experimentation, at least for the initial screening of potential insecticides.

Evaluation of a home visiting and befriending scheme for young mothers: Newpin.

Befriending schemes to support families with young children are now widespread in the United Kingdom. One such scheme, Newpin, which aims to prevent child abuse is described. Evaluation compared those recruited to befriend as well as those befriended. Assessments at baseline and 6 months' follow-up comprised interviews with mothers, developmental assessments of the children and observation of mother-child interaction using video tapes. All assessments were conducted in the home and similar measures were applied to a control sample in an area where a Newpin scheme was planned. Newpin recruits, whether befrienders or befriended had high rates of adverse early experiences, but depression was more common amongst those referred for befriending. Approximately a third did not sustain involvement in the scheme, but those that did were more likely to have had training as volunteers and mental health problems of more than 2 years' duration.

The impact of maternal depression in young children.

An urban working class sample of depressed mothers with two-yr-old children was compared with a control group of non-depressed mothers using maternal interviews, home observation of mother/child interaction, and developmental assessment of the children. Measures were repeated after six months. Children of depressed mothers showed more emotional and behavioural disturbance and delay in expressive language development. Depressed mothers had more past and current adverse experiences, particularly in close relationships. In general depressed mothers were less responsive to their children and less able to sustain social interaction: their children were more often distressed, but there was a big variation in quality of mother/child interaction within the depressed group.

An autoradiographic study of the cell proliferation during involution of the rat pancreas.

After ligation of the rat pancreas, DNA synthesis in centoacinar cells and cells of the intercalary ducts, proximal and distal to the ligature, was suppressed for about 18 hr. This preceded a large increase in thymidine labelling of the nuclei of these cells. The increase in the thymidine indices was much greater and more prolonged in the distal pancreas where duct-like structures were formed that replaced the acini. DNA synthesis in acinar cells proximal to the ligature was suppressed for 36 hr preceding an increase in the thymidine indices much smaller than that in the duct cells. DNA synthesis in acinar cells distal to the ligature ceased and the acinar cells progressively died. We propose that the pancreas is composed of proliferative units, each comprising acinar cells, centroacinar cells and intercalary ducts, which react as a whole when acinar cell loss occurs in pathological processes.

The efficacy of some acaricides against screw-worm fly larvae.

Thirteen acaricides used for control of cattle tick in Queensland were evaluated for their potential in the chemical control of the screw-worm fly, Chrysomya bezziana. Laboratory evaluations and in vivo tests using artificially infested cattle were made in Papua New Guinea. Most of the acaricides caused some mortality of screw-worm larvae in infested cattle and in laboratory tests. Acaricides of the organophosphorous, carbamate and organophosphorous/synthetic pyrethroid groups showed reasonable activity against screw-worm fly, but the amidines were less effective.

Involution of the pancreas after ligation of the pancreatic ducts. I: a histological study.

A detailed histological study of pancreatic involution after ligation of the pancreatic duct is reported. Cytological evidence acinar cell damage is evident from the beginning and loss of zymogen granules occurs early. The acini diminish in size rapidly from the 36th hour to the 3rd day. The cell deletion appears to occur in 2 phases with apparently different characteristics. The first phase appears to be related to the initial cell damage. The letter phase is more general but of uncertain nature. Considerable proliferation of cells occurs in the ducts resulting, with the few remaining acinar cells, in duct-like structures which themselves undergo involution. The duct changes therefore are not passive but involve continued cell proliferation and cell death over a long period.

Mercuric chloride-induced tubulonecrosis in the rat kidney: the recovery phase.

Regeneration of the renal tubules in rats, after death of the cells of the pars recta caused by a dose of 1.5 mg HgCl2/kg, was examined by histological and radioautographic methods. The tubules regenerated from surviving squamoid cells at both ends of the necrotic segments, which often appeared to arise from epithelial cells that had cast off superficial "dead cytoplasm" to leave the basal parts containing the nucleus in a rim of cytoplasm. The tubular epithelium was reconstituted between the 2nd and 5th days by proliferation and sliding extension of the squamoid cells along the tubules and predominantly from the distal end of the necrotic segments where the cell proliferation was extremely active. Inflammation in reaction to the dead cells was insignificant. Although the majority of tubules regenerated in a regular fashion some degree of anomalous epithelial proliferation occurred in patches, predominantly in the junctional area between the pars recta and the loops of Henle and perhaps most frequently in reference to those nephrons with superficial and mid-cortical glomeruli. The exuberant proliferation led to scattered epithelial growths projecting into the lumen of the tubules, and there was evidence that these obstructed the discharge of necrotic debris. Tubular collapse and atrophy leading to the formation of small scars followed, more often affecting the short looped nephrons.

Mercuric chloride-induced renal tubular necrosis in the rat.

A light microscopic study of the renal tubulonecrotic lesion in rats given a small dose of HgCl2 is described. The changes consist of a rapidly developing vacuolation of the cytoplasm with loss of basophilic staining within 4 h that leads to cell breakdown, fragmentation and dissolution by 48 h. Nuclear changes appear to set in later. Permanent patchy fibrotic lesions were found in the kidneys at 10 days. The animals pass a large amount of urine of low osmolarity, low Na+, K+ and Cl- for a period of 3 days accompanied by an increased water intake. Nevertheless there appeared to be no water or ionic imbalance between daily inputs and outputs. Blood urea levels were greatly increased for 3 days, but did not return to normal by the 10th day.

Repeated partial hepatectomy as a promoting stimulus for carcinogenic response of liver to nitrosamines in rats.

Partial hepatectomy 24 h before a single i.p. dose of dimethylnitrosamine, diethylnitrosamine or ethylmethylnitrosamine increased the carcinogenic response in the liver of rats as determined by the number of tumours and the number of "focal proliferations" produced. Secondly, in rats given a single i.p. dose of diethylnitrosamine, 3 partial hepatectomies 5, 10 and 15 weeks after dosing the animals increased the carcinogenic response in the liver. The stimulus of repeated partial hepatectomy therefore appears to act as a "promoting agent" for liver carcinogenesis, that is if the single dose of diethylnitrosamine is regarded as an "initiating agent" in the terms of the two-stage hypothesis.

Influence of carbon tetrachloride on induction of tumours of the liver and kidneys in mice by netrosamines.

Mice were given a single dose of nitrosolimethylamine, nitrosoliethylamine or nitrosomethylethylamine and the yield of tumours and related lesions in the livers determined 12 months later. A hepatonecrotic dose of CCl4 24 or 48 h before the nitrosamines, increased the yields of hepatocellular tumours and proliferative foci in the livers, whereas when given 60 h before the nitrosamines there was no significant effect.

The binding of ethyl carbamate to DNA of mouse liver in vivo: the nature of the bound molecule and the site of binding.

Ethyl carbamate, labelled at C1 with 14C, bound in vivo to liver DNA of intact and partially hepatectomised mice. Isotope (18O) enrichment was not detected in the oxygen of liver DNA of mice injected with [18O] ethyl carbamate, C2H5--18O--CO--NH2. This suggests that it was the ethyl group and not the ethoxy group which bound to DNA. Chromatographic analysis of acid hydrolysates of liver DNA from mice treated with [1-14C] ethyl carbamate provided no evidence of alkylation or other form of binding to purine or pyrimidine bases. On relatively mild acid hydrolysis the alkyl group remained bound to the "apurinic acid" fraction, while more vigorous hydrolysis lead progressively first to its separation as highly ionisable hydrophilic non-volatile compounds and then to its loss as a volatile compound. DNAase I followed by phosphodiesterase hydrolysis also split off the 14C-containing group as a volatile compound. The volatile compound was identified as ethanol. These results suggest that the alkyl group was bound as an ester to a phosphate group in the DNA chain. Results with DNA from partially hepatectomised mice did not differ from those with DNA from intact mice.

Carcinogenesis by carbamic acid esters and their binding to DNA.

The tumor-initiating potency of three simple alkyl carbamates and mono-N-substituted ethyl carbamates was examined in Hall strain mice. The binding of 14C-labeled carbamates of DNA was measured in Crackenbush mice. Ethyl carbamate was the most potent carcinogen for the epidermis, liver, and lung, followed by its N-alkyl derivatives. Methyl carbamate was without effect but n-propyl and n-butyl were possible carcinogens. The ethyl esters bound to a greater extent to DNA in liver and skin than the methyl, n-propyl, and n-butyl esters and only this binding persisted. A preliminary application of croton oil increased the yield of skin tumors but not of liver or lung tumors. It also increased the binding of the alkyl carbamates to DNA in skin, the increase being greatest with ethyl carbamate. The binding persisted longer in treated than in non-croton oil-treated mice.

Partial hepatectomy and toxicity of dimethyl-nitrosamine and carbon tetrachloride, in relation to the carcinogenic action of dimethylnitrosamine.

The yield of tumours in the liver of rats was increased when dimethylnitrosamine was given 1, 6 or 12 h after partial hepatectomy and still further increased if it was given after an interval of 24-72 h. The increase was greater after two-thirds than after one-third hepatectomy. An increase in the number of kidney tumours was also found. Microsomal DMN-demethylase activity was depressed after partial hepatectomy for up to 6 days in mice and rats. The LD50 of DMN on the other hand was decreased for 3 days, after which it returned to normal. The extent of liver necrosis produced by DMN was increased at 6 and 24 h after partial hepatectomy but was within the usual range at longer intervals. These results suggest that prolonged exposure of the tissues to DMN after partial hepatectomy played a significant role in the development of liver tumours as well as those in the kidney, in addition to the role of regeneration of the liver, and that the relative roles were still to be elucidated.